This a competitive proposal that is based on two sets of preliminary data. In the first the PI has shown that cultured adrenocortical cells release into the supernatant one or more ouabain-like inhibitors of the Na, K pump. In the second, the PI has developed an HPLC-assisted quantitive reverse transcription-polymerase chain reaction (QRTPCR) method to quantitate mRNA levels in small amounts of tissue. Based on these, he is proposing two aims. Aim 1 is to utilize several modern analytical techniques to characterize the inhibitor (s) of the Na, K pump released by adrenal Y1 cells. This will done via preliminary HPLC purification of relatively large amounts of supernatant medium which will be followed by tandem mass spectrometry and if the material reaches sufficient purity, by high resolution NMR. Selected chemical modifications of high purity material may also be used to aid in identification of the aforementioned inhibitor. Aim 2 is to quantitate the levels of mRNA via the aforementioned QRTPCR method in defined segments of the rat nephron under different levels of dietary sodium intake.